1. Field of the Invention
The present invention relates to a urogenital sinus derived growth inhibitory factor, ps20. More specifically, the present invention relates to uses of the factor and to the amino acid and nucleotide sequence of said factor. The present invention also relates to antibodies which bind to said factor.
2. Description of the Prior Art
Epithelial differentiation patterns are induced by stromal cells in most eukaryotic tissues including lung, breast, stomach, skin, eye, and prostate gland. In prostate development, ductal morphogenesis and epithelial acini differentiation result from stromal induction. In heterotypical tissue-tissue recombinants, mesenchyme from fetal urogenital sinus (anlagen of the prostate gland) induces bladder epithelial cells to undergo ductal morphogenesis and differentiation to a prostatic epithelial phenotype, capable of expressing prostate specific proteins.
Prostate ductal morphogenesis is characterized by a stromal-induced epithelial cell proliferation. Following this, epithelial cytodifferentiation (to the secretory phenotype) is correlated with a cellular quiescence which also requires stromal interaction. In most cells, differentiated gene expression is associated with a reduced cell proliferation. In cell culture, growth stimulatory activities (to epithelial cells) have been observed in the conditioned medium from prostate stromal cells, including bFGF and NGF-like factors. Progress has been made in the identification of keratinocyte growth factor (KGF) as a likely candidate stromal-derived factor. KGF expression is restricted to stroma and is androgen regulated in the prostate. KGF mediates in part the stromal induction of seminal vesicle epithelium proliferation. TGF-.beta.1 and TGF-.beta. receptors are negatively regulated by androgen in the prostate. TGF-.beta.s are expressed in development, however, their role in prostate development is not clear. Growth inhibitory activities secreted from prostate stromal cultures have been reported by our laboratory group and others, not attributed to known inhibitory factors including the TGF-.beta.s. This growth inhibitory activity is attributable to the urogenital derived sinus growth inhibitory factor (UGIF)/ps20 protein, which also induces protein synthesis and alters phenotypic morphology of target epithelial cells. As with all other growth regulatory proteins, ps20 is not specific to prostate, but is also expressed in mesenchymal and smooth muscle cells. The developmental pattern of prostatic ductal morphogenesis followed by epithelial differentiation likely involves the timed expression of a variety of positive and negative growth regulatory factors.
Studies with rat and human prostatic smooth muscle cell lines show androgen-stimulated proliferation with physiological concentrations (5-10 mM) of androgen. These observations together indicate urogenital sinus mesenchyme and adult smooth muscle cells likely to express genes fundamental to stromal-epithelial interactions in the prostate gland.
Progress has been limited in identification of stromal-derived regulatory proteins and their mechanisms due to technical difficulties in the isolation and culture of androgen responsive stromal cell lines, difficulties in the biochemical analysis of secreted or extracellular matrix proteins, and the relative unavailability of tissue-specific stromal cell cDNA libraries.
Benign prostatic hyperplasia (BPH) and prostate cancer are disorders of prostatic epithelial growth and differentiation. BPH disorders are perhaps most relevant to stromal-epithelial interactions. BPH initiates from localized stromal cell proliferation. The initiation of BPH has been termed a "reawakening" of the inductive potential of the prostate stroma and a spontaneous reversion of the stroma to an embryonic state. Accordingly, the abnormal proliferation of stromal cells in the periurethral region can induce the ingrowth and abnormal formation of acini from adjacent epithelial cells.
During prostate carcinogenesis, carcinoma progression patterns involve stromal-epithelial interactions. Prostatic carcinoma is typified by progression from an androgen responsive state to an androgen insensitive state which no longer responds to anti-androgen therapy. Some evidence exists to suggest that progression to androgen insensitivity results from altered gene expression in stromal cells. In the Dunning rat prostate carcinoma, the type of stroma can induce the adjacent epithelium to exhibit exon switching of FGF receptors (FGFRc2 IIIb to IIIc) which imparts androgen insensitive proliferation to these epithelial cells. Dunning tumor prostate carcinoma cell proliferation was inhibited by 7-fold when recombined with normal seminal vesicle or urogenital sinus mesenchyme. The recombined carcinoma cells showed an alteration in phenotypic morphology. When recombined with normal mesenchyme, carcinoma cells exhibited a tall, columnar cell shape, typical of a differentiated secretory epithelium as compared to the typical squamous/cuboidal undifferentiated phenotype in wild-type Dunning tumor. In this regard, it is of interest that smooth muscle is absent from Dunning prostatic tumor. In addition, the pattern of carcinoma formation can be influenced by the origin of the associated stromal cells. Recombination of bladder transitional cell carcinoma with normal urogenital sinus mesenchyme resulted in the formation of a glandular adenocarcinoma phenotype typical of prostate. Tissue-tissue recombination studies to produce prostatic tumors in mice requires transformation of mesenchyme (with myc and ras) to produce prostatic adenocarcinoma typical of the human phenotype. Conversely, the inoculation of fibrosarcoma tumorigenic stromal cells with non-tumorigenic normal epithelial cells into nude mice resulted in a mixed carcinoma-fibrosarcoma. Together these studies indicate prostatic carcinoma epithelium is responsive to the stromal environment and that progression and overall phenotype of prostate carcinoma is dependent to some degree on stromal interaction. It follows that key proteins involved in mechanisms of stromal-epithelial interactions will be of significance to the study of prostate proliferation diseases.
Balance of protease and protease inhibitor function is involved in modeling of tissues, extracellular matrix (ECM) compositions, and growth factor activation processes. Proteases play a significant role in embryogenesis, extracellular matrix modeling/remodeling and in tumorigenesis involving abnormal proliferation, promotion of tumor invasion, and formation of metastasis. It is well-established that metalloproteinases are overexpressed in most neoplastic diseases including breast cancer, colon cancer, neuroblastomas, and prostate cancer. Cysteine proteases including Cathepsins B and D are elevated in many cancer metastases, including prostate cancer. Significant to tumor progression, due to their induced cascade of effects are the plasminogen activator (PA) proteases. Plasminogen activators are serine proteases which convert inactive plasminogen to the active form, plasmin. Plasmin in turn exhibits broad proteolytic trypsin-like effects on ECM components including glycoproteins, proteoglycans (including heparin and heparin-sulfates), and gelatins. Plasmin also activates a variety of EMC bound growth factors from latent to active forms including the TGF-.beta.s. The urokinase-type
and tissue-type PA have been the most extensively studied PAs. Urokinase PA is primarily involved in tissue modeling-remodeling activities, whereas tissue PA is most active in blood clot lysis.
Since plasminogen is present in all tissues and fluids, local effects of plasmin are mediated by local expression of PA. Urokinase PA is secreted as an inactive pro-form which binds with high affinity to a membrane-anchored specific receptor where it is cleaved to the active form and remains (on cell surface) for several hours. On the cell surface, urokinase PA has a focal effect resulting in local acceleration of plasmin activation by approximately 40-fold. Plasmin activity is elevated in the focal environment to the cell surface expressing active urokinase. Focal plasmin effects degradation of ECM and activates metalloproteinases (procollagenases, prostromelysin, elastase). Accordingly, secretion of small amounts of urokinase PA results in a focal plasmin cascade to effect a spectrum of other enzymes and factors. Urokinase PA is inhibited by PA inhibitors (PAIs) which are serine protease inhibitors. Local actions of PAs (and other proteases) have been implicated in a wide array of developmental processes through highly regulated mechanisms. PAs, PAIs, and proteases are each regulated by hormones and growth factors.
In addition to functions in development, urokinase PA is elevated in most tumor metastases. Elevated urokinase PA leads to down stream activation of proteases and growth factors with increased tumor invasion, increased tumor volume, and increased cell proliferation rate. In the prostate, the study of proteases and inhibitors have focused primarily on carcinoma progression. PA activity is higher in prostate carcinoma than in normal tissue and the urokinase PA form is primarily associated with progression. Urokinase PA is elevated in prostate bone metastasis relative to primary tumor site. Urokinase PA is overexpressed in Dunning, Nobel, Lobund-Wistar, and Fisher-334 prostatic tumors. Moreover, urokinase PA is the predominant PA secreted by the PC-3 and DU-145 human prostatic carcinoma cell lines and these cell lines exhibit the urokinase PA cell surface receptor. Our studies have used the PC-3 cell line to identify and purify ps20 secreted from fetal urogenital sinus mesenchymal cells. Metastasis of PC-3 in nude mice was blocked by mutated urokinase PA or urokinase PA receptor blocking antibodies.
Direct evidence shows growth inhibition of cancer cells by urokinase PA inhibitors and other protease inhibitors. A synthetic urokinase PA inhibitor (p-aminobenzamidine) inhibited the progression of DU-145 human prostate carcinoma in SCID mice and cell proliferation in culture in a dose-dependent manner (64% decreased tumor volume). The protease inhibitor actinonin inhibited mammary tumor progression (both non-metastatic and metastatic types) in collagen gels. Batimastat, a matrix metalloproteinase inhibitor inhibited organ invasion in lung (72% decrease in tumor volume) of two human colon carcinomas. In human prostate, decreased expression of acid cysteine proteinase inhibitor (ACPI) (cathepsin inhibitor) was observed in BPH tissue relative to normal. No expression of ACPI was found in human prostatic adenocarcinoma tissue. Accordingly, balances of proteases and protease inhibitors likely affects proliferation in human BPH and carcinoma.
U.S. Pat. No. 5,196,334, incorporated by reference herein, describes the isolation and partial characterization of urogenital sinus derived growth inhibitory factor, UGIF (ps20). However, the amino acid and nucleotide sequence of ps20 has not heretofore been described. Additionally, antibodies to ps20 have also not heretofore been described.